SUMMARY The overarching objective of this Phase I project is to determine the feasibility of developing both focused and broad toxicity metabolomic assays for three neural development associated windows of susceptibility (WOS) for human development. Epidemiological and laboratory studies have identified numerous chemicals suspected to be developmental neurotoxicants. These range from heavy metals that have been in our environment for millennia to newer chemicals used to limit fire damage and kill unwanted pests. New and more informative metabolomic assays will provide key pathway analysis information that when associated with adverse outcome pathways at the cellular and animal level will lead to better predictive metabolomic in vitro assays for candidate neurotoxicants. We will use our innovative and unique suite of commercialized cell type neural progenitor (hNP1TM), neuronal (hN2TM) cells and a new astrocyte line (hAstroProTM) in individual metabolomic assays. No other commercial or nonprofit organization can provide these proprietary highly enriched cells. These cells span the approximate time of neural tube formation in human development. In the second aim we will also utilize a new coculture format for neuronal (hN2TM) and glia (hAstroProTM) to be used to determine metabolomic profiles of cell types in more complex co-culture systems. The cell to cell interaction is more representative of the cellular and tissue composition of the developing central nervous system than individual cells or co-culture systems that physically isolated populations of cells such as trans well cocultures. Specifically, we plan to use an innovative rapid label free cell separation system developed by ArunA Biomedical and collaborators after the coculture is exposed to a toxicant. This process isolates neurotoxicant exposed hN2TM and hAstroProTM cell fractions for metabolomic analysis. We believe that the selected neurotoxicants will generate WOS-specific metabolome signatures that are linked to other key events in the adverse outcomes, including cell/organ function and development, in a dose-dependent manner. This provides the foundation for developing more focused metabolomic arrays and assays that can be used with pluripotent cell derived neural cells and directly reprogrammed hNP1 cells from a diverse genetic population. This project will lead to a Phase II proposal where resulting commercial products includes an array of metabolomic assays. At the end of Phase II we will have developed neural pathway specific metabolomic assays and global assays that use simple and complex neural developmentally relevant cell cultures. ArunA will provide this as a service (cell assays) and outsource the metabolomic analysis and in some cases lease the cell assays to specific customers.